Enzyme-substrate interactions have long been recognized as representing an extreme expression of structural complementarities in biological chemistry. An enzymatic reaction can be broken down into two main aspects. The first involves the diffusion-controlled formation of the encounter complex, and the second involves the appropriate structural and dynamical arrangements of the enzyme domains as dictated by the reaction chemistry. It is the long-term goal of the PIs research program to investigate enzyme-small molecule (inhibitor, substrate, or cofactor) interactions in order to unravel the structural and dynamical aspects of its reaction. This proposal aims to study the hydride transfer mechanism involved in DHPR. Dehydropteridine reductase (DHPR) catalyses the NADH-mediated reduction of quinonoid dihydrobiopterin (qBH2) to yield tetrahydrobiopterin (BH4). BH4 functions as an essential cofactor, and its absence leads to depletion in the brain of precursors of catecholamine and serotonin neurotransmitters. Regulation of DHPR became of interest when a new form of hyperphenylalanineanemia (atypical phenylketonuria) associated with a defect in BH4 recycling was discovered. In the light of other new emerging functions, there is a growing need to completely understand how DHPR works. The overall objective of the investigation is to understand the structural changes that occur when DHPR binds cofactor, substrate or inhibitor. Using advanced fluorescence and vibrational techniques, we will identify catalytically important bonds as well as detect major protein conformational changes that may accompany binding and catalysis. We will also look at the dynamical aspects of the binding and catalytic events using fluorescence-based temperature-jump studies. The results will be married to the solved X-ray crystal structures of DHPR to gain insights into the mechanism of enzyme action. In particular, we would like to address the mechanistic role of the cofactor and the conserved Cys residue. Since DHPR belongs to a superfamily of biologically important short-chain dehydrogenases/reductases (SDRs), it is hoped that the gathered results could provide some mechanistic details useful for studying SDRs. SDRs have diverse functions. Some are good pharmacological targets while others are important in controlling the cellular availability of a hormone receptor ligand. There are also those that have essential role in growth factors or nutrient synthesis. Understanding the enzymology of DHPR at the molecular level will play a part in realizing the untold potential for drug design, enzyme regulation, and enzyme engineering.

Public Health Relevance

Enzyme-substrate interactions have long been recognized as representing an extreme expression of structural complementarities in biological chemistry. Basic research geared towards understanding the inner workings of an enzyme system, like dihydropteridine reductase (DHPR), is important if cures for the diseases caused by a malfunctioning or deficient enzyme are to be found. Regulation of DHPR became of interest with the discovery of atypical phenylketonuria, a neurological disorder, associated with a defect in tetrahydrobiopterin recycling.